Characterization and mRNA expression profile of the TbNre1 gene of the ectomycorrhizal fungus Tuber borchii

Curr Genet (2009) 55:59–68 DOI 10.1007/s00294-008-0222-x R ES EA R C H A R TI CLE Characterization and mRNA expression proWle of the TbNre1 gene of the ectomycorrhizal fungus Tuber borchii Michele Guescini · L. Stocchi · D. Sisti · S. Zeppa · E. Polidori · P. Ceccaroli · R. Saltarelli · V. Stocchi Received: 28 July 2008 / Revised: 11 November 2008 / Accepted: 16 November 2008 / Published online: 30 December 2008  Springer-Verlag 2008 Abstract This study focuses on the cloning and characterization of the major nitrogen regulator element from the ectomycorrhizal fungus Tuber borchii, TbNre1. Sequence analysis of the predicted protein and complementation experiments in Neurospora crassa demonstrated that the cloned gene is orthologous to areA/nit-2 gene. Transcriptional expression investigations by real-time RT-PCR showed TbNre1 up-regulation in the presence of nitrate or in the absence of nitrogen during free-living mycelium growth. On the contrary, TbNre1 mRNA levels remained at basal values in the presence of preferred nitrogen sources like ammonium and glutamine. Furthermore, TbNre1 mRNA was found to be up-regulated during T. borchii and T. platyphyllos interaction. All these data suggest that the regulatory protein TBNRE1 could play a major role in regulating N metabolism genes of T. borchii in the free living Communicated by U. Kües. Electronic supplementary material The online version of this article (doi:10.1007/s00294-008-0222-x) contains supplementary material, which is available to authorized users. M. Guescini · L. Stocchi · S. Zeppa · E. Polidori · P. Ceccaroli · R. Saltarelli · V. Stocchi (&) Department of Biomolecular Science, Institute of Biological Chemistry “G. Fornaini”, University of Urbino “Carlo Bo”, Via SaY, 2, 61029 Urbino, Italy e-mail: vilberto.stocchi@uniurb.it M. Guescini e-mail: michele.guescini@uniurb.it D. Sisti Department of Human, Environmental and Nature Science, University of Urbino “Carlo Bo”, Campus ScientiWco Sogesta, Loc Crocicchia, 61029 Urbino (PU), Italy mycelium and in T. borchii–T. platyphyllos interaction. Finally, the possible role of the transcription factor TBNRE1 in the induction of proteases and virulence-like genes, necessary in ectomycorrhizal establishment, was also discussed. Keywords Tuber borchii · Ectomycorrhizae · Gene regulation · Nitrogen limitation · Zinc-Wnger transcription factor Introduction Filamentous fungi utilize a wide range of nitrogen (N) compounds as their sole N source. To ensure a suYcient supply of N for growth in rapidly changing environments, fungi possess a large array of catabolic genes dedicated to the utilization of various secondary N compounds, such as nitrate, purine and amides. The expression of speciWc genes corresponding to the available N source provides a selective advantage to fungi. In particular, eco-physiological studies have demonstrated the ability of plant colonizing fungi to sustain the N nutrition of their host plants (Martin et al. 2001). Through the formation of specialized symbiotic structures, called ectomycorrhizae, mycorrhizal fungi dramatically expand the eVective surface area for nutrient soil exploration, broaden the range of exploitable inorganic and organic N sources, and improve competition with other soil micro-organisms (Read 1999). Indeed, recent data have shown that ectomycorrhizal fungi have an important role in mobilizing N from well-decomposed organic matter (Lindahl et al. 2007). The hyphal network permeating the soil might express a wide diversity of proteolytic enzymes as is the case of the ectomycorrhizal fungus Laccaria bicolor in which a large number of secreted proteases have 123 60 been identiWed, conWrming the ability of this fungus to use N of animal origin (Martin et al. 2008). Central to this mutualistic interaction is the exchange of plant-derived carbohydrates with ready-to-use organic N sources provided by the fungus (Buscot et al. 2000). In order to gain insight into how ectomycorrhizal mutualism works, we need to understand which forms of N are being taken up by the fungus and transferred to the plant, how they are being transported, and what regulates this uptake and transport system. Previous biochemical studies have reported that the ectomycorrhizal fungus T. borchii signiWcantly contributes to N nutrition of its host plant modulating the expression of N induced genes in response to diVerent N status and to the ectomycorrhizal symbiosis (Guescini et al. 2003, 2007; Montanini et al. 2002, 2003, 2006a, b; Pierleoni et al. 2001; Vallorani et al. 2002). In the saprophytic and pathogenic ascomycete fungi, A. nidulans and N. crassa, N-induced response is governed by the regulatory circuit which is controlled in parallel by major and minor (pathway-speciWc) regulatory genes (Caddick et al. 1994; Marzluf 1997). Fungal major N regulatory proteins are positive-acting transcription factors similar to those belonging to the mammalian GATA family (Merika and Orkin 1993; Scazzocchio 2000). All members of this family carry a DNA-binding domain made of a single Cys-2/Cys-2 zinc Wnger followed by an adjacent basic region that recognizes the consensus GATA motif in promoter sequences of target genes. In response to nutrients in their environments, N metabolism is tightly regulated by positively or negatively acting transcription factors in the model organisms Neurospora crassa and Aspergillus nidulans (Marzluf 1997). The positively acting transcription factors (GATA factors) are expressed when fungi sense that preferred N sources, such as ammonium and glutamine, are limited, repressed when these chemicals are abundant. During N starvation, GATA factors activate the transcription of genes involved in N catabolic pathways by binding to promoter sequences (GATAboxes). Negative transcription factors act when in the fungal environment an adequate N is present. This process is well studied in non-mycorrhizal fungi; however, much less is known about ectomycorrhizal fungi such as T. borchii or whether N starvation prevails during plant infection. Recent studies have described the up-regulation of the T. borchii nitrate transporter (TbNrt2) and nitrite reductase (tbnir1), which were found to be induced by nitrate, but also through a nitrate independent derepression mechanism triggered by N starvation (Guescini et al. 2007; Montanini et al. 2006b). A similar trend of regulation was found in the ectomycorrhizal basidiomycete Hebeloma cylindrosporum in which the nitrate transporter, the nitrate and nitrite reductase, transcription is repressed by ammonium and active 123 Curr Genet (2009) 55:59–68 under N deprivation or in the presence of a secondary N source such as nitrate (Jargeat et al. 2000; Jargeat et al. 2003). In this study, we report the cloning of the N regulatory gene T. borchii Nitrogen regulator element 1 (TbNre1), which is homologous to nit-2 and area, from T. borchii. The functional equivalence of this gene to nit-2 was demonstrated by the transformation of the TbNre1 gene into a nit-2 mutant strain of N. crassa obtained by repeat-induced point (RIP) mutagenesis and the checking of the ability of the transformants to utilize nitrate. Furthermore, the transcriptional regulation of this gene during the establishment of the ectomycorrhizal symbiosis was investigated. Materials and methods Growth of T. borchii mycelium and T. borchii–T. platyphyllos ectomycorrhizae Tuber borchii Vittad. mycelia (strain MYA-1018), used to assess TbNre1 gene expression levels, were grown for 20 days in modiWed Melin-Norkrans nutrient solution (MMN) (Molina 1979) containing 3 mM ammonium phosphate [(NH4)2HPO4] as N source. The mycelia, kept in a growth chamber at 24°C in the dark with no agitation, were transferred to MMN liquid medium containing one of the following N sources: 3 mM ammonium phosphate, 3 mM potassium nitrate and 3 mM L-glutamine and growth was continued for 5 more days. T. borchii–T. platyphyllos symbiotic interactions were obtained in a 135-mm plate using the same medium reported in Pierleoni et al. (2001), except that agarose was added and the sole N source was 3 mM potassium nitrate. DNA and RNA isolation Genomic DNA was isolated from 1-month-old cultures of T. borchii mycelia following the protocol described by Zeppa et al. (2001). Total RNA was isolated from free-living mycelia and the ectomycorrhizae were obtained as described above using the RNeasy Plant Mini kit (Qiagen) according to the manufacturer’s instructions. The Wnal concentration and quality of the RNA samples were estimated both spectrophotometrically and by agarose gel electrophoresis. Cloning of the major N regulator element TbNre1 from T. borchii Two degenerate oligonucleotides, NRE1 (5⬘-TGTACNA AYTGYTTYACNCA-3⬘) and NRE2 (5⬘-TTCTTPATNA CPTCNGTYTT-3⬘), were used to prime a PCR with genomic Curr Genet (2009) 55:59–68 DNA from T. borchii. The ampliWcation reaction was carried out in a total volume of 25
l, with 200 ng of genomic DNA, 1£ reaction buVer, 2 mM MgCl2, 100
M dNTPs, 50 pmol of each primer and 0.5 U of AmpliTaq DNA polymerase (Perkin Elmer). The mixture was incubated for 5 min at 94°C and then subjected to 35 cycles of 1 min at 95°C, 1 min at 42°C and 30 s at 72°C, with a Wnal cycle of 15 min at 72°C. The ampliWcation products of about 136 bp were fractionated on a 3% agarose gel and ligated into the pGEM-T vector (Promega). The ligated fragment was sequenced and used as a homologous probe (pZF) to screen a lambda EMBL4 T. borchii genomic DNA library (a gift of Prof. Viotti A., Istituto Biosintesi Vegetali, CNR, Milan). Southern hybridization analysis Genomic DNA samples for gel blot analysis (10
g each) were digested with the enzymes HindIII BamHI, and KpnI and then electrophoresed on a 0.8% agarose gel. DNA was blotted onto positively charged Hybond N+ (ver. 2.0) nylon membranes (Amersham Life Science), in accordance with the manufacturer’s instructions, and hybridized in phosphate buVer (Sambrook and Russel 2001) with the pZF or A7-A9 fragments, which were labelled with 32P using the RediPrime labelling kit (Amersham Life Science). The Wnal post-hybridization wash was carried out in 15 mM NaCl, 1.5 mM trisodium citrate (0.1X·SSC) and 0.1% SDS at 65°C. Cloning, sequencing and sequence analyses DNA sequencing was performed by gene-walking on both strands of the T. borchii genomic library clones. Database searches were performed using the BLAST2 program (Altschul et al. 1997) and multiple alignment of protein sequences using CLUSTAL W (Thompson et al. 1994). IdentiWcation of conserved motifs was carried out by searching the BLOCKS (HenikoV and HenikoV 1994) and PRINTS (Attwood and Beck 1994) databases using IDENTIFY (Stanford University, http://dna.stanford.edu/identify/). Introns within the TbNre1 coding sequence were localised by sequencing cDNA segments. Using pairs of speciWc oligonucleotides deduced from the genomic sequence (data not shown), overlapping cDNA fragments (ranging in size over 0.3–1.0 kb) were ampliWed by reverse transcriptionpolymerase chain reactions (RT-PCR), using 1
g of total RNA extracted from T. borchii mycelia grown on a nitratecontaining medium. Reverse transcription with the Omniscript reverse transcriptase (Qiagen) and PCR reactions using Taq DNA polymerase (Qiagen) were carried out following standard protocols. AmpliWed cDNA segments were 61 cloned in the pGEM-T vector (Promega) and sequenced. The cloned gene fragments were sequenced in both directions using the ABI PRISM BigDye Terminator CycleSequencing Ready Reaction kit (Perkin-Elmer), according to the manufacturer’s instructions, and the ABI PRISM 310 Genetic Analyzer (PE Applied Biosystems). The TbNre1 gene appears in the GenBank database under the accession number EU917069. N. crassa transformation Neurospora crassa transformation was carried out according to Ballario et al. (1996); brieXy, 7-day-old conidia of the nit-2 RIP-350 strain were electroporated using the Gene Pulser II (Bio-Rad) in the presence of 1 M sorbitol and 1.2
g of plasmidic DNA construct pMYX2–TbNre1 (containing the TbNre1 full-length open-reading frame under the control of a strong promoter region). Transformants were selected on Vogel’s N-free medium supplemented with 20 mM ammonium and Benomyl 2
g/ml. Subsequently, the resistant transformants were tested for their ability to grow in the presence of 20 mM nitrate as the sole N source. Quantitative real-time PCR (qRT-PCR) One microgram of DNaseI (Ambion)-treated total RNA was reverse transcribed as already described by Guescini et al. (2007). The samples used for the reverse transcription were the RNA extracted from T. borchii mycelia grown as described above and from T. borchii–T. platyphyllos ectomycorrhizae. T. borchii 18S rRNA (tb18S) was used as an internal reference gene. SpeciWc primers for TbNre1 (TBNRE1F: 5⬘-CCTTCGCCAACGGTATTC-3⬘ and TBNRE1R. 5⬘-TGAACACAAGCCACCACTT-3⬘) and tb18S (TB18SF: 5⬘-ACTGGTCCGGTCGGATCTT-3⬘ and TB18SR: 5⬘-TTCAAAGTAAAAGTCCTGGTTCCC-3⬘) were designed to amplify under the same cycling conditions and procedure reported in Guescini et al. (2007). PCR was performed in a Bio-Rad iCycler iQ Multi-Color RealTime PCR Detection System. The speciWcity of the ampliWcation products obtained was conWrmed by examining thermal dissociation plots and by sample separation in a 3% DNA agarose gel. The amount of the target transcript was related to that of the reference gene using the method described by PfaZ (2001). Each sample was tested in triplicate by quantitative PCR, and data obtained from at least three independent experiments were used to calculate the means and standard deviation. The Kruskal–Wallis test (non parametric ANOVA) was used to identify signiWcant diVerence in expression levels in time course and N-induction experiments. The Mann–Whitney U-test was used to compare the medians of TbNre1 mRNA levels between 123 62 Curr Genet (2009) 55:59–68 free-living mycelia and ectomycorrhizal tissues. All the results were considered signiWcant if P values were < 0.05. Amino acid analysis Mycelia and ectomycorrhizae were homogenized in HClO4 to precipitate all proteins, and the suspensions thus obtained were centrifuged at 14,000 rpm for 10 min. Supernatants were neutralized with K2CO3 and aliquots of 10 and 20
l used to determine the amino acid content as described by De Bellis et al. (1998). Pellets were then resuspended in 0.5 N NaOH, and total proteins were evaluated as reported by Saltarelli et al. (1998). Results Cloning and characterization of the T. borchii nitrogen regulator element 1 gene In order to synthesize a homologous probe for the TbNre1 gene we performed an aminoacid sequence alignment of the major N regulator element from the following fungi: N. crassa, A. nidulans, Penicillum chrysogenum and Magnaporthe grisea. From this analysis it was possible to identify a highly conserved region, the zinc-Wnger domain, that was used to design two degenerate primers, NRE1 and NRE2, which ampliWed a 136-bp PCR fragment (called pZF) from T. borchii genomic DNA. The nucleotide sequence obtained from this amplicon was used to search the protein databanks and showed high homology to the major N regulator element areA (identity 97%), nmc (identity 97%), clnr1 (identity 97%) and nit-2 (identity 95%) from A. nidulans (Kudla et al. 1990), Penicillum roquefortii (Gente et al. 1999), Colletotrichum lindemuthianum (Pellier et al. 2003) and N. crassa (Fu and Marzluf 1990), respectively (S1). This analysis clearly demonstrated that the cloned PCR product encoded for the N regulatory element from the ectomycorrhizal fungus T. borchii. pZF fragment was used as a probe to screen a lambda EMBL4 T. borchii genomic library, and two hybridizing plaques were detected and puriWed. Digestion of the inserts of these phages with the enzymes EcoRI, BamHI and XhoI resulted in four fragments, and the sequencing and contig of these clones allowed us to obtain the complete sequence of the TbNre1 gene (Fig. 1). BlastX and BlastN searches combined with alignments between TbNre1 gene and TbNre1 cDNA sequences allowed us to identify the putative coding region of TbNre1 (Accession No. EU917069). The sequence analysis revealed an encoding region of 2,793 bp interrupted by two small introns of 72 and 64 bp, which were found in the amino-terminus of the predicted protein and placed in 123 Fig. 1 Strategy used to obtain the full-length genomic sequence of the T. borchii TbNre1 gene. The inserts of the diVerent plasmids pB13, pE37, pB23 and pB7, retrieved from the clone fTbNre1 of the lambda EMBL4 T. borchii genomic library, are positioned above the genomic library clone and have been used in the sequencing reactions. The restriction map of the clone is reported at the top. B: BamHI; E: EcoRI. The empty boxes represent introns present in the TbNre1 gene. The pA7-A9 DNA fragment used as probe to identify the TbNre1 gene in Southern blot experiment (see Fig. 2) is also shown Fig. 2 Southern blot analysis. 32 P-labelled pA7-A9 (see Fig. 1) was used as a probe on genomic DNA from T. borchii mycelia digested with the enzymes HindIII (lane 1), BamHI (lane 2) and KpnI (lane 3). The migration positions of DNA size markers are indicated on the left conserved positions compared to nit-2 (Fu and Marzluf 1990), nut-1 (Froeliger and Carpenter 1996) and areA-Gf (Tudzynski et al. 1999) (S2). In order to investigate the TbNre1 genomic organization, Southern blot analysis was performed; T. borchii genomic DNA was digested with restriction enzymes BamHI, KpnI and HindIII which did not cut the probe sequence, and hybridised with the clone A7-A9 (see Fig. 1). Under highand low-stringent conditions, this probe produces only one hybridisation signal in the digested DNA, indicating the presence of a single copy of TbNre1 in the T. borchii genome (Fig. 2). The TbNre1 gene encodes for a 931-amino-acid polypeptide. Its amino acid sequence could be aligned with Curr Genet (2009) 55:59–68 63 those of other fungal N regulator elements, with identity scores of 40, 38, 37 and 36% for comparisons between T. borchii and A. nidulans, Penicillium roquefortii, N. crassa and Colletotrichum lindemuthianum N regulation elements, respectively. Sequence alignments with known N regulation element protein sequences identiWed three functional domains within the TbNre1 deducted protein sequence: the N-terminal domain with unknown function (R135–M145), the central-zinc Wnger domain highly conserved (P697–S751) and the C-terminal domain which show the two conserved motives VIPIAAAPPK (C1) and EWEWLTMSL (C2) (S3). Complementation of a N. crassa nit-2 mutant with the TbNre1 In order to ascertain that the genomic clone TbNre1 encodes for the major N activator protein from T. borchii, the pTbNre1 construct was used to transform a nit-2 RIP350 mutant strain of N. crassa and the transformants were then tested for their ability to grow in nitrate medium. The N. crassa nit-2 mutant was used because T. borchii TbNre1 mutants were not available. Transformation was integrative and Southern blot analysis showed that all transformants contained at least one copy of the plasmid pTbNre1 (S4). The transformation eYciency was 10–15 transforming colonies per microgram of used DNA; the empty pMYX2 vector was used as a negative control. Four of these N. crassa transformants were tested for their ability to grow on minimal medium supplemented with ammonium or nitrate. Unlike the nit-2 RIP-350 mutant strain, the N. crassa nit-2 mutants complemented with TbNre1 gene were able to grow in minimal medium containing nitrate as the sole N source (S4). However, the growth of these transformants was not as extensive as the growth of the N. crassa wildtype strain. Hence, we can conclude that the T. borchii TbNre1 gene is a functional homologue of the N. crassa nit-2 gene. Fig. 3 TbNre1 gene transcriptional regulation in diVerent nitrogen regimens. mRNA accumulation has been quantiWed by Real-time RTPCR using tb18S as reference gene as described in materials and methods section. Mycelia were cultured for 20 days in MMN medium containing 3 mM (NH4)2HPO4 as nitrogen source (Control), and after this period of growth the mycelia were shifted for 1, 2, 3 and 5 days to MMN medium containing: 3 mM ammonium phosphate (NH4+), 3 mM glutamine (GLN), 3 mM potassium nitrate (NO3¡), and no nitrogen source (STV). The data represent average values of three independent measurements (§SD). Statistical diVerences were assessed by the Kruskal-Wallis test, the results were considered signiWcant if P values were <0.05.* Ctrl versus NO3¡ and § Ctrl versus STV conditions starvation the TbNre1 mRNA levels were found to be consistently high. On the contrary, TbNre1 mRNA levels remained at basal values in the presence of preferred N sources like ammonium, and glutamine (Fig. 3). Because the 3⬘-UTR region was implicated in the areA mRNA stability (Morozov et al. 2001; Platt et al. 1996), a homolog of the TbNre1, the comparison analysis of the 3⬘-UTR of areA, nre (the major nitrogen regulator from P. chrysogenum) and TbNre1 were performed. This sequence analysis showed a considerable identity only between areA and nre, while the sequence of the TbNre1 3⬘-UTR was less conserved (S5). The TbNre1 mRNA levels in T. borchii mycelia grown under diVerent nitrogen sources Evaluation of the TbNre1 mRNA levels in T. borchii–T. platyphyllos ectomycorrhizal tissue The real-time RT-PCR technique was used to investigate the N conditions that induce the expression of the TbNre1 gene. Mycelia were grown for 20 days in MMN medium containing 3 mM ammonium phosphate as N source and then transferred for 1, 2, 3 and 5 days to 3 mM ammonium phosphate, 3 mM glutamine, 3 mM potassium nitrate or in N-free medium (starvation), respectively. As shown in Fig. 3, we found a strong TbNre1 mRNA up-regulation following transfer to either nitrate-supplemented medium or N limited conditions. In the case of nitrate induction, the TbNre1 mRNA up-regulation was transient, while in N We assessed the transcriptional regulation of the TbNre1 gene during the process of T. borchii–T. platyphyllos interaction. The host plant and T. borchii mycelia were grown in the same plate and the interaction between the two symbionts was obtained in a medium containing 3 mM potassium nitrate. This growth system led both to the production of secondary roots by the host plant and a high mycelial growth up to Wll the plate. During this ectomycorrhizal interaction, a threefold induction of the TbNre1 mRNA was found as compared to control mycelia (Fig. 4). 123 64 Curr Genet (2009) 55:59–68 glutamine, since they are considered as crucial signalling molecules in N metabolism regulation. As reported in Table 1, the levels of the amino acids under study did not show signiWcant changes among mycelia transferred to diVerent nitrogen sources, with the sole exception of the mycelia grown in N-free medium where the levels of aspartic acid, asparagine, glutamic acid and glutamine were not detectable. Finally, we were not able to Wnd diVerences in amino acid levels in either the extramatrical mycelium associated with T. platyphyllos or in uninoculated mycelia. Discussion Fig. 4 Real-time RT-PCR quantiWcation of the TbNre1 mRNA levels in T. borchii free-living mycelia and after T. borchii–T. platyphyllos symbiotic interactions. a Representative image showing symbiotic T. borchii–T. platyphyllos interactions obtained. b TbNre1 mRNA accumulation was quantiWed by Real-time RT-PCR using tb18S as reference gene. The data represent average values of three independent measurements (§SD). Statistical diVerences were assessed by the Mann–Whitney U-test, *the results were considered signiWcantly diVerent if P values were <0.05 Amino acid analysis in T. borchii free-living mycelium and in T. borchii–T. platyphyllos ectomycorrhizal tissue In order to further investigate the contribution of the fungal symbiont to the host plant N nutrition, we measured the levels of free amino acids in free-living mycelia transferred to diVerent nitrogen sources, free-living mycelia growth in interaction medium (uninoculated mycelia) and in extraradical mycelia associated with T. platyphyllos. In particular, we assayed aspartic acid, glutamic acid, asparagine and 123 The essence of ectomycorrhiza function is the bidirectional exchange of soil-delivered nutrients, mainly phosphorus and N, delivered by the fungus, for plant-derived carbohydrates (Read 1999). Despite the global importance of mycorrhizal fungi and their potential in agriculture, our knowledge in this Weld is still limited. The uptake of mineral nutrients from soil by plants is greatly aided by mutualistic associations with mycorrhizal fungi. In addition to beneWting plants by aiding phosphorus uptake from the soil (Bücking 2004; Ducic et al. 2008), the importance of mycorrhizal fungi in acquiring N for the plant has been convincingly demonstrated for ectomycorrhizal fungi (Chalot et al. 2006; Guescini et al. 2003, 2007; Martin et al. 2001; Montanini et al. 2002, 2003; Vallorani et al. 2002). The observed up-regulation of several genes involved in nutrient assimilation reXects the intense metabolite Xuxes occurring between the symbiotic partners and reveals a complex interplay of fungal and plant transporter activities. This further highlights the need for Weld studies to elucidate the importance of mycorrhizal fungi to plant N nutrition. One of the factors thought to favour the establishment of ectomycorrhizal symbiosis is N limitation (Buscot et al. 2000; Lilleskov et al. 2002). The observation that many genes, induced under N-limiting conditions, possess several GATA sequence motives (canonical N regulatory elements), in their promoter regions, together with the fact that these genes are often up-regulated during ectomycorrhiza establishment, prompted us to study the transcriptional regulation of the major N regulator element from the ectomycorrhizal fungus T. borchii. Several lines of evidence demonstrate that the cloned gene, TbNre1, is a major N regulatory gene of T. borchii, orthologous to areA/nit-2 gene. First, sequence comparisons of the TBNRE1 protein with NIT-2, NUT-1 and AREA-GF showed high levels of amino acid sequence similarities throughout the protein. Second, the highest degree of identity was observed within the zinc Wnger domain. Like NIT-2 and AREA, the TBNRE1 protein contains a conserved C-X2- Curr Genet (2009) 55:59–68 65 Table 1 Determination of free amino acid levels in the T. borchii freeliving mycelium grown in the presence of diVerent nitrogen sources, in free-living mycelium grown in interaction medium (uninoculated Source of material mycelia) and in the extramatrical T. borchii mycelium associated with T. platyphyllos roots Amino acid content (
mol/
g of protein) Aspartic acid Asparagine Glutamic acid Glutamine NH4+ 0.045 § 0.01 1.17 § 0.3 0.39 § 0.1 0.65 § 0.04 Glutamine 0.065 § 0.03 1.3 § 0.1 0.52 § 0.06 0.77 § 0.02 NO3¡ 0.035 § 0.04 1.0 § 0.1 0.41 § 0.12 0.5 § 0.1 STV Not detectable Not detectable Not detectable Not detectable Uninoculated mycelia 0.047 § 0.03 0.95 § 0.1 0.37 § 0.07 0.49 § 0.15 Ectomycorrhizae 0.034 § 0.05 0.85 § 0.1 0.38 § 0.03 0.59 § 0.2 Free-living mycelium transferred in Mycelia were grown for 20 days in the presence of MMN medium containing 3 mM phosphate ammonium as sole nitrogen source and then transferred to diVerent nitrogen sources for 5 days. Free-living mycelium and ectomycorrhizae were grown in interaction medium as reported in materials and methods section C-X17-C-X2-C zinc-Wnger motif with an adjacent downstream basic region characteristic of the GATA family of transcription factors (Crawford and Arst 1993; Marzluf 1997). Furthermore, the C-terminal domain shows the two highly conserved motives VIPIAAAAPPK (C1) and EWEWLTMSL (C2). These two motives are important in the transcriptional activation mechanism where they seem to be involved in the binding between the NIT-2 protein with its inhibitor NMR. Third, the comparison of growth between the nit-2 RIP350 mutant strain of N. crassa and the nit-2 RIP-350 mutant strain complemented with TbNre1 gene in ammonium or nitrate, as the sole N source, conWrmed that the TbNre1 gene acts as a global nitrogen regulatory gene. These data are further corroborated by cross experiments in which the bidirectional promoter TbNrt2-tbnr1 (Guescini et al. 2007) was found to respond to nitrogen supply in N. crassa as host organism (B. Grimaldi, personal comunication). Subsequently, we investigated the nitrogen conditions that induce the expression of the TbNre1 gene in the T. borchii mycelia. Real-time RT-PCR experiments showed a basal TbNre1 expression in the presence of primary N sources such as ammonium or glutamine, while an up-regulation was found in T. borchii mycelia grown in media containing nitrate, as the sole N source, or in the absence of N supplies. These data suggested that N derepression (i.e. the absence of primary N sources) is suYcient to trigger the TbNre1 expression. N derepression could also act to stabilize TbNre1 mRNA, as reported for the areA/nit-2 homologs (Morozov et al. 2001; Tao and Marzluf 1999). Though the sequence analysis of the TbNre1 3⬘-UTR region failed to show obvious conserved motives, this post-transcriptional regulation mechanism could exist in TbNre1 and should be properly investigated in future studies. Moreover, the availability of the TbNre1 gene allowed us to investigate the regulation of this key transcription factor of N metabolism during ectomycorrhiza establishment. DiVerential expression of the fungal nitrite reductase gene (Hc-nir) in mycorrhizae and in the extra-radical mycelia was found in the H. cylindrosporum/Pinus pinaster association. The Hc-nir transcription levels were always far higher in the free-living mycelia exposed to an N-free medium than transcript accumulation during symbiotic interaction (Bailly et al. 2007). On the contrary, in this study, we demonstrated that TbNre1 mRNA is up-regulated during T. borchii and T. platyphyllos interaction, which is in agreement with previous studies showing the induction of the TbGS (Montanini et al. 2003), TbNrt2, tbnr1 and tbnir1 genes, in the T. borchii–T. platyphyllos ectomycorrhizae (Guescini et al. 2003, 2007). All these data suggest that the regulatory protein TBNRE1 could play a major role in regulating N metabolism genes of T. borchii in free-living mycelium and in T. borchii–T. platyphyllos interaction. In an attempt to conWrm this hypothesis, we evaluated the levels of free amino acids in free-living mycelia, grown in diVerent nitrogen sources and during interaction with T. platyphyllos roots. Unfortunately, this analysis did not show any signiWcant diVerence between free-living mycelia and ectomycorrhizae. However, on the basis of this result, the existence of the transfer of N compounds from fungus to plant cannot be excluded. Indeed, recent studies have hypothesized a direct transfer of NH4+ in other mycorrhizal models (Chalot et al. 2006; Selle et al. 2005). Furthermore, the exchange of N within ectomycorrhizal tissues probably occurs at the symbiotic interface and involves small Xux of compounds that cannot be detected if related to the whole extramatrical mycelium. Nevertheless, the very low levels of amino acids found in mycelia grown in nitrogen deprivation, a condition in which the TbNre1 reaches its highest expression levels, further demonstrates the key role of amino acids, and in particular of glutamine, in the transcriptional regulation of TbNre1. 123 66 In addition, other genes, not directly involved in N metabolism but strongly inXuenced by N starvation, have been previously identiWed in T. borchii mycelia (Montanini et al. 2006a; Soragni et al. 2001). One of these, the TbSP1 gene, was also found up-regulated in the ectomycorrhizal tissue (Miozzi et al. 2005). A shared feature highlighted in the promoter regions of the TbNrt2, tbnr1, tbnir1 and TbSP1 genes is the presence of putative binding sites for the global N regulator AREA/NIT-2 (Guescini et al. 2007; Montanini et al. 2006a, b; Scazzocchio 2000). Furthermore, several studies have suggested that starvation is one of the signals controlling the expression of genes involved in the pathogenicity of various plant microbial pathogens (Snoeijers et al. 2000). These genes exhibit GATA motives in their promoter sequences which are the core recognition sequences for the binding of AREA/NIT-2-like proteins. For example, the Cladosporium fulvum avr9 gene, which is expressed in planta during the infection process, is induced in vitro under nitrogen starvation. The regulation of this gene is under the control of the global N regulator NRF1 and is also observed in planta (Perez-Garcia et al. 2001). Likewise, the induction of the M. grisea mpg1 gene in vitro under nitrogen starvation is under the control of both the suspected additional N regulators, NPR1 and NPR2 (Lau and Hamer 1996), and there is no indication that either the avr9 gene or the mpg1 gene is directly involved in N metabolism. Like promoters of the A. nidulans, structural genes that are regulated by AREA (Scazzocchio 2000), the promoter region of avr9 contains AREA binding sites (Snoeijers et al. 1999). Thus, induction of avr9 results from binding of a trans-acting nitrogen regulatory protein. Compatible with this scenario, we might hypothesize that during ectomycorrhiza establishment one of the key transcription factors activated in this process is TBNRE1, which in addition to activating the utilization of diVerent N sources, might be involved in the induction of proteases and virulence-like genes necessary in the ectomycorrhizal interaction. This hypothesis is supported by recent data showing that ectomycorrhizal fungi have an important role in mobilizing nitrogen from well-decomposed organic matter (Lindahl et al. 2007). Indeed, the hyphal network permeating the soil might express a wide diversity of proteolytic enzymes, as is the case of the ectomycorrhizal fungus L. bicolor, in which a large number of secreted proteases have been identiWed, conWrming the ability of this fungus to use N of animal origin. These proteases may also have a role in developmental processes, because the expression of several secreted proteases is up- or down-regulated in ectomycorrhizal root tips (Martin et al. 2008). Furthermore, it has recently been reported that GATA transcription factors control the expression of the secreted aspartic protease, SAP2, which is required both for the utilization of alternative N sources and for virulence (Dabas and Morschhäuser 2008). 123 Curr Genet (2009) 55:59–68 The role of the TbNre1 gene during ectomycorrhizal interaction merits further investigation. 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